The recent characterization of CD25+ T regulatory (CD25 Treg) cells has underscored the importance of these cells in modulating immune responses in a broad range of disease states. CD25 Treg cells are a subset of regulatory T cells with an anergic phenotype that suppress immune responses in an antigen specific fashion by a poorly understood mechanism. To date, the only unique genetic marker in CD25 Treg cells is the transcription factor, Foxp3. Studies from our laboratory have demonstrated a link between GRAIL, an E3 ubiquitin ligase that is necessary for the development of CD4 T cell anergy in vivo, and CD25 Treg cells. We hypothesize that GRAIL is important for the development and function of naturally occurring and induced CD25 Treg cells. Several lines of evidence support this hypothesis: 1) GRAIL is expressed in naturally occurring CD25 Treg cells at levels 10-fold greater than CD25- CD4 T cells, 2) a tolerizing immunization in vivo leads to the induction of long-lived CD25 expressing antigen-specific tolerized T cells. Gene expression analysis of these cells reveals that GRAIL mRNA is upregulated (700-fold increase vs. CD25- tolerized; 100-fold increase vs, naturally occurring CD25 Treg cells). Moreover, GRAIL expression is linked with Foxp3 expression strongly suggesting a suppressor phenotype in this induced tolerized population. As an initial step to understanding the role of GRAIL in CD25 Treg cell suppressor function, we demonstrate that enforced expression of GRAIL in an antigen-specific T cell line is sufficient to convey a suppressor phenotype in vitro. The specific aims of this research proposal are: 1) To establish the role of GRAIL in naturally occurring CD25 Treg cell development and function by generating bone marrow chimeric mice expressing GRAIL, a dominant negative form, or GRAIL RNAi construct 2) To determine if GRAIL is upregulated in activated suppressor cells by functionally characterizing the induced CD25+ GRAIL expressing tolerized cells and establishing the kinetics of GRAIL expression in activated suppressor cells.